Project Summary PTEN, encoding phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is a tumor suppressor gene mutated in a variety of human cancers. However, unlike other tumor suppressor genes, even small perturbations in PTEN activity can promote tumorigenesis suggesting it is highly regulated. Post-translational regulation of PTEN can affect protein stability, localization, and conformation as well as phosphatase activity, and in many cases the effects are cell type specific. Our lab has observed that PTEN translated from the long 3'UTR isoform is significantly less active in the PI3K pathway than PTEN translated from the short 3'UTR isoform, and that the expression of 3'UTR isoforms varies across tissue and cell types. The goal of the proposed study is to elucidate the molecular mechanism of 3'UTR-dependent regulation of PTEN activity to augment our understanding of disease mechanisms in PTEN dysregulated cancers. The proposal has two specific aims. Aim 1 will investigate the biological consequence of 3'UTR-dependent PTEN regulation. Since there is no observed difference in PTEN protein levels in cells that predominantly express the long 3'UTR isoform compared to cells that predominantly express the short 3'UTR isoform, the hypothesis is that alternative isoform usage must effect either subcellular localization, homo-dimerization, or phosphatase activity. Exclusive shRNA-mediated knockdown of the long 3'UTR isoform will be used to generate cells that predominantly express the short 3'UTR, which will be compared to control cells that express both isoforms equally. The cell lines will be immunostained for endogenous PTEN and subcellular localization can be compared. They will also be used to immunoprecipitate protein translated from primarily the short 3'UTR and protein translated from both isoforms to assess how dimerization and phosphatase activity is affected by alternative 3'UTR isoform usage. In Aim 2, these cell lines will be used to identify protein factors that specifically interact with the long 3'UTR isoform. The hypothesis is that the long 3'UTR of PTEN recruits a protein that forms a complex with newly translated PTEN, thus repressing its activity in the PI3K pathway. Long 3'UTR-specific protein interaction partners will be identified by mass spectrometry and the information obtained in Aim 1 will be leveraged to select candidates to validate for their role in regulating PTEN protein. shRNA will be used to knockdown candidate interaction partners. If a candidate is required for 3'UTR-dependent PTEN regulation, then shRNA-mediated knockdown of this candidate will restore activity of PTEN translated from the long 3'UTR isoform to that of PTEN translated from the short 3'UTR isoform.